HD-domain proteins constitute a novel superfamily of metalloenzymes that counts presently more than 37,000 members in all three domains of life. Though there are generally annotated as as (phospho)hydrolases their functions are mostly unknown. Less than a decade ago, a novel diiron HD enzyme involved in the catabolism of inositol and associated with type I diabetes mellitus, namely myo-inositol oxygenase was demonstrated to carry out a radically different reaction using molecular oxygen to afford activation of its substrate. The only recently identified HD enzyme PhnZ, was also shown to follow the paradigm of MIOX, employing oxygen for the conversion of an organophosphonate to phosphate by marine microorganisms. The mechanism and structure of the reactive intermediates are presently unknown, but their mechanistic striking similarity to other nonheme Fe enzymes involved in the biosynthesis of antibiotics, invoke questions about the function of these enzymes and their possible implications on human health and environment. For this purpose, the first part of the project will focus on the characterization of PhnZ. This entails a combination of spectroscopic, structural, redox and activity studies so as to establish the modus operandii of such novel oxygenases. The substrate-free and bound forms of the enzyme will be studied so as to obtain for the first time combined structural and electronic information about the 'on' and 'off' reactive states for which crystallographic information has been extremely challenging, how substrate or inhibitors tune and affect the properties of the active site of the enzyme. This information will set the grounds for the discovery of compounds that can activate and inhibit these enzymes, therefore providing powerful control over their function. In addition to, the characterization of downstream events in the chemical reactions will likely establish the common strategy that specific nonheme Fe enzymes (mononuclear or dinuclear) adopt to carry out difficult and environmentally important reactions. The second part of the project aims at mapping the catalytic landscape of (dinuclear) HD domain enzymes, discovery of new functions and identifying the type and the role of metals in modulating specific activities (hydrolysis vs oxygenation). For this purpose, on the basis of phylogenetic analysis new attractive protein targets of unknown function have been identified. Selected protein will be overexpressed and purified. These will be spectroscopically characterized with a combination of EPR, Mssbauer, crystallographic and NMR techniques. A profile of their activities will be established by screening activities for specific substrates and mass spectrometry methods. Presently there are a handful of HD domain enzymes implicated in immunoresponse, such as restriction factors for HIV-1 or nucleotidases attacking viral nucleotides that have come into the scientific focus. Their function is not completely understood, whereas the presence of one or two metals is not known whether it is functional, structural or co-catalytic. This work will begin during the K99 funding period and will continue during the independent phase and will attempt to study these enzymes and draw the molecular background of their function (hydrolytic vs oxygenation). The long-range purpose of this second phase of the project is to establish on the basis of bioinformatics, crystallographic, mutagenesis and activity studies the determinants directing distinct functions within the HD superfamily that will ultimately lead to the identification new antiviral factors and therapeutic agents as well as the discovery of novel oxygenases implicated in chemically difficult small molecule transformations.